Mixotrophic chain elongation with syngas and lactate as electron donors

Abstract Feeding microbial communities with both organic and inorganic substrates can improve sustainability and feasibility of chain elongation processes. Sustainably produced H2, CO2, and CO can be co‐fed to microorganisms as a source for acetyl‐CoA, while a small amount of an ATP‐generating organic substrate helps overcome the kinetic hindrances associated with autotrophic carboxylate production. Here, we operated two semi‐continuous bioreactor systems with continuous recirculation of H2, CO2, and CO while co‐feeding an organic model feedstock (lactate and acetate) to understand how a mixotrophic community is shaped during carboxylate production. Contrary to the assumption that H2, CO2, and CO support chain elongation via ethanol production in open cultures, significant correlations (p < 0.01) indicated that relatives of Clostridium luticellarii and Eubacterium aggregans produced carboxylates (acetate to n‐caproate) while consuming H2, CO2, CO, and lactate themselves. After 100 days, the enriched community was dominated by these two bacteria coexisting in cyclic dynamics shaped by the CO partial pressure. Homoacetogenesis was strongest when the acetate concentration was low (3.2 g L−1), while heterotrophs had the following roles: Pseudoramibacter, Oscillibacter, and Colidextribacter contributed to n‐caproate production and Clostridium tyrobutyricum and Acidipropionibacterium spp. grew opportunistically producing n‐butyrate and propionate, respectively. The mixotrophic chain elongation community was more efficient in carboxylate production compared with the heterotrophic one and maintained average carbon fixation rates between 0.088 and 1.4 g CO2 equivalents L−1 days−1. The extra H2 and CO consumed routed 82% more electrons to carboxylates and 50% more electrons to carboxylates longer than acetate. This study shows for the first time long‐term, stable production of short‐ and medium‐chain carboxylates with a mixotrophic community.

PVC tubing (Tygon ® LMT-55, Saint-Gobain Performance Plastics Nagano, Japan) was used for all gas and the remaining liquid lines.
The flexible gas reservoir had a maximum volume of 22 L and the rigid headspace volume of the system was 2.3 L. The gaseous substrate was replenished when one of the following conditions was met: all CO was consumed, the gas reservoir volume was less than 10 L, or the gas bag volume was bigger than 20 L due to net gas production. During replenishment, the empty gas reservoir was filled with 17 L syngas mixture (32% H2, 32% CO, 16% CO2, 20% N2) with 480 mL He injected as a tracer gas and, when applicable, ethylene. Ethylene was used as a methanogenesis inhibitor auxiliary to CO. Since we have previously observed acclimatization of methanogens to ethylene after long exposure (Baleeiro et al. 2021), 2 kPa ethylene (ca. 420 mL) was only used when deemed necessary (between days 47 and 92). This way, methanogenic activity was kept low, with a maximum of 4.4% CH4 accumulating in the gas phase and an average CH4 production rate of 11 mL L -1 d -1 , corresponding to less than 1% of the total organic carbon fed. Air contamination of the systems was monitored by measuring the N2 content of the gas phase. The details and calculations for monitoring air contamination have been described previously (Baleeiro et al. 2022). Except for two unintended air contamination events during overnight operation on days 229 (Reactor 2) and 251 (Reactor 1), the reactors were kept largely anoxic with an average ORP of −386 ± 73 mV and an average O2 intrusion rate of 13 mL L -1 d -1 .

CARBON FIXATION AND ASSUMPTIONS FOR ESTIMATING THE SOURCE OF CARBON
DIOXIDE EMISSIONS. The rate of carbon fixation rfixed (in C mmol L -1 d -1 ) was calculated as described in Equation (1).
In order to estimate whether the CO2 emissions were due to lactate decarboxylation or CO oxidation to CO2, CO2 production rates were first corrected by considering the CO2 that was consumed for CH4 and the CO2 that got washed-out in the medium.
First, we assumed that every for every CH4 molecule produced, one CO2 molecule was consumed. The dissolved CO2 concentration in the medium was estimated to be 11.8 mM CO2, leading to CO2 wash-out rates of 0.843 mmol CO2 L -1 d -1 (HRT 14d) and 1.18 mmol CO2 L -1 d -1 (HRT 10 d). This concentration of CO2 in the medium was obtained using Henry's law and the carbonate system equilibria. Assuming 32°C, pH 6.0, and 28.7 kPa CO2 (average CO2 partial pressure). Further assumptions for estimating the source of CO2 emissions were: 1) The amount of decarboxylated lactate (i.e. lactate that emits one CO2 molecule) can be estimated by subtracting the production rates of all odd-chain compounds from the consumption rates of lactate.
3) Lactate decarboxylation is a more favorable route for CO2 emission than CO oxidation.
The corrected CO2 production rates were subtracted from the amount of decarboxylated lactate to obtain an estimate of the source of CO2 emissions. If the resulting value was positive, it was interpreted as CO2 emissions from lactate decarboxylation that were abated by CO2 fixation. If the resulting value was negative, it was interpreted as evidence for CO2 being formed via CO oxidation. NaOH, were dissolved in deionized water under aerobic conditions. The resulting solution was then made anoxic by stirring for at least three hours in an anaerobic chamber. Then, the anoxic solution was transferred into a 2-L borosilicate glass bottle with a two-port cap. The ports of the bottle were kept sealed by crimped silicon tubes during transportation and storage in the anaerobic chamber. Afterwards, the glass bottle with the solution was sterilized by autoclaving (121ºC for 20 minutes) and stored inside the anaerobic chamber. Vitamins and cysteine hydrochloride were added to the medium in the anaerobic chamber just before connecting the bottle to the reactor system's feed pump. These solutions stemmed from concentrated solutions of vitamins (previously sterilized with a 0.2 µm syringe filter) and cysteine hydrochloride (previously sterilized by autoclaving). Originally, the medium had a pH value of 2.6 (with acetate) or 3.9 (acetate-free). The pH was automatically adjusted to 6.0 by adding 4 M NaOH after being pumped into the reactor. During operation, the salinity of the broth depended on the amount of NaOH added to keep a stable pH value. Therefore, the salinity depended on the total amount of carboxylates in the broth and was estimated to be between 13 and 28 g NaCl eq. L -1 . propionate, and 69 mg L -1 n-butyrate), whereas the broth of the enrichment culture was rich in various carboxylates (9.13 g L -1 acetate, 2.53 g L -1 n-butyrate, 4.62 g L -1 n-caproate, and 1.21 g L -1 n-caprylate).
ASV CLUSTERING. Among the 25 most abundant ASVs found in this dataset, the ones assigned to Clostridia (i.e. 22 ASVs) were aligned de novo using the library Decipher for R (Wright 2015). The aligned sequences were clustered using the Neighbor Joining model with help of the libraries Phangorn (Schliep et al. 2017) and Ape (Paradis et al. 2004) for R. To identify poor alignment due to short sequences (ASVs were ~400 base pairs long), the sequences were additionally aligned with a reference E. coli sequence using the web-based tool SILVA Alignment, Classification and Tree Service (Quast et al. 2013). All alignments had a score of 98 or more.      However, these two genera were only transiently detected in the community, as Caproiciproducens, Clostridium sensu stricto 12, Oscillibacter, and Eubacterium became more abundant from day 75.
Between days 75 and 110, the n-caproate concentrations settled at around 2.5 g L -1 and the most distinctive feature between the two reactors was a higher propionate concentration coinciding with higher abundance of Acidipropionibacterium, the putative propionate producer.
In comparison to the first 61 days of fermentation with the diverse community (Reactor 1, Fig. 1), the period between days 110 and 148 in Reactor 1 (feeding interval 4 d) showed net acetate consumption (13.6 emmol L -1 d -1 ), a decrease in gas consumption by 40% (to 45.2 emmol L -1 d -1 or 554 mL L -1 d -1 of H2 + CO), and an increase in elongation to n-caproate by about 47% (to 45 emmol L -1 d -1 ) (Reactor 1, Fig. S7A). For the following experiments, both reactors were operated with a feeding interval of 3.5 days (feeding twice per week) for ease of operation.
Interrupting acetate supply greatly increased carbon fixation rates in both reactors but decreasing HRT did not show a clear effect on fixation rates. Once both reactors were operating without acetate supply, a carbon fixation rate of 31.0 C mmol L -1 d -1 was seen in Reactor 1 with an HRT of 14 d and 28.0 C mmol L -1 d -1 was seen in Reactor 2 with an HRT of 10 d during the same period (Fig. 2B). Net carbon fixation was majorly due to CO consumption since CO2 was a net product under all conditions. Net consumption of CO2 can likely be achieved, if a syngas mixture with less CO is to be used.
The decrease of HRT from 14 to 10 d (i.e. a 40% dilution rate increase) corresponded to a 41% increase in the production rates of C≥4 carboxylates (from 111 to 156 emmol L -1 d -1 , Fig. 2A) pointing out that the system could be optimized for higher rates without loss of selectivity to longer-chain carboxylates.